-
Current Protocols in Chemical Biology. 03/2013;
-
[show abstract]
[hide abstract]
ABSTRACT: Microvesicles (exosomes) are important mediators of intercellular communication, playing a role in immune regulation, cancer progression, and the spread of infectious agents. The biological functions of these small vesicles are dependent on their composition, which is regulated by mechanisms that are not well understood. Although numerous proteomic studies of these particles exist, little is known about their glycosylation. Carbohydrates are involved in protein trafficking and cellular recognition. Glycomic analysis may thus provide valuable insights into microvesicle biology. In this study, we analyzed glycosylation patterns of microvesicles derived from a variety of biological sources using lectin microarray technology. Comparison of the microvesicle glycomes with their parent cell membranes revealed both enrichment and depletion of specific glycan epitopes in these particles. These include enrichment in high mannose, polylactosamine, α-2,6 sialic acid, and complex N-linked glycans and exclusion of terminal blood group A and B antigens. The polylactosamine signature derives from distinct glycoprotein cohorts in microvesicles of different origins. Taken together, our data point to the emergence of microvesicles from a specific membrane microdomain, implying a role for glycosylation in microvesicle protein sorting.
Journal of Proteome Research 08/2011; 10(10):4624-33. · 5.11 Impact Factor
-
Consortium of Functional Glycomics; 07/2011
-
Avital Gaziel-Sovran,
Miguel F Segura,
Raffaella Di Micco,
Mary K Collins,
Douglas Hanniford,
Eleazar Vega-Saenz de Miera,
John F Rakus,
John F Dankert,
Shulian Shang,
Robert S Kerbel,
Nina Bhardwaj,
Yongzhao Shao,
Farbod Darvishian,
Jiri Zavadil,
Adrian Erlebacher, Lara K Mahal,
Iman Osman,
Eva Hernando
[show abstract]
[hide abstract]
ABSTRACT: To metastasize, a tumor cell must acquire abilities such as the capacity to colonize new tissue and evade immune surveillance. Recent evidence suggests that microRNAs can promote the evolution of malignant behaviors by regulating multiple targets. We performed a microRNA analysis of human melanoma, a highly invasive cancer, and found that miR-30b/30d upregulation correlates with stage, metastatic potential, shorter time to recurrence, and reduced overall survival. Ectopic expression of miR-30b/30d promoted the metastatic behavior of melanoma cells by directly targeting the GalNAc transferase GALNT7, resulted in increased synthesis of the immunosuppressive cytokine IL-10, and reduced immune cell activation and recruitment. These data support a key role of miR-30b/30d and GalNAc transferases in metastasis, by simultaneously promoting cellular invasion and immunosuppression.
Cancer cell 07/2011; 20(1):104-18. · 25.29 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: β-O-N-acetyl-D-glucosamine (O-GlcNAc) is a post-translational modification involved in a plethora of biological systems ranging from cellular stress to insulin signaling. This modification shares many hallmarks with phosphorylation, including its dynamic cycling onto a host of proteins such as transcription factors, kinases, and phosphatases, and regulation of cellular functions, including cell signaling. Herein, we report the development of an improved genetically based O-GlcNAc FRET sensor and compartmentalized targeted variants for the characterization of the spatiotemporal dynamics of O-GlcNAc. During serum-stimulated signal transduction, rapid increases in O-GlcNAc activity were observed at both the plasma membrane and the nucleus, with a concomitant decrease detected in the cytoplasm. These findings suggest the existence of compartment specific dynamics for O-GlcNAc in response to signal-inducing stimuli, pointing to complex regulation of this modification. In addition, inhibition of the PI3K pathway by wortmannin abolished the O-GlcNAc response, suggesting that the activity observed is modulated downstream of the PI3K pathway. Taken together, our data argues that O-GlcNAc is a rapidly induced component of signaling and that the interplay between O-GlcNAc and kinase signaling may be more akin to the complex relationship between kinase pathways.
Journal of Biological Chemistry 02/2011; 286(8):6650-8. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Herein we describe the orientation of GST-tagged lectins on NHS-activated slides via a one-step deposition of the protein and a glutathione (GSH) scaffold. This technology overcomes the need for a premade GSH-surface to orient GST-tagged proteins, enabling us to rapidly expand the analytical capacity of lectin microarrays through addition of oriented lectins, while maintaining lectin diversity.
Molecular BioSystems 01/2011; 7(7):2114-7. · 3.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The mammalian cell surface is rich with carbohydrate polymers involved in a diversity of biological recognition events. Dynamic alterations of surface glycans mediate cell-cell communication in the immune system and host specificity of bacterial and viral pathogens. In addition, altered surface glycosylation has been implicated in disease progression of many cancers and may serve as important new targets for therapeutics. Despite the importance of glycosylation, the systematic analysis of sugars, i.e., glycomics, has lagged behind the well-studied disciplines of genomics and proteomics. This deficiency is due in part to the unique analytical challenges presented by glycans and the overwhelming diversity of sugars in nature. New microarray technologies have provided a high-throughput methods with which to probe the glycome. Our laboratory has pioneered a shown ratiometric two-color lectin microarray method that rapidly evaluates differences in the glycosylation of mammalian cells. Herein, we present a detailed protocol of our lectin microarray methodology for the differential analysis of mammalian glycomes.
Methods in molecular biology (Clifton, N.J.) 01/2011; 671:117-31.
-
[show abstract]
[hide abstract]
ABSTRACT: The cell surface is covered with a myriad of carbohydrates that form a complex matrix of oligosaccharides. Carbohydrate recognition plays critical roles in pathogenesis, trafficking, and differentiation. Lectin microarray technology presents a novel platform for the high-throughput analysis of these structurally diverse biopolymers. One drawback of this technology has been limitations imposed by the commercially available plant lectins used in the array. Not only are a majority of these plant-derived proteins glycosylated, which can complicate glycomic analysis, but they also differ in activity and availability. Our lab has recently introduced recombinant lectins to enhance the stability and scope of our lectin panel. Herein, we provide a detailed procedure for the expression of bacterially-derived lectins and their application to a recombinant lectin microarray.
Methods in molecular biology (Clifton, N.J.) 01/2011; 723:67-77.
-
[show abstract]
[hide abstract]
ABSTRACT: The emerging roles for post-translational modifications in the regulation of cellular function have turned the spotlight on
glycosylation. Given the prevalence of protein and lipid glycosylation, it has become imperative to create and utilize new
tools to study these critical biopolymers. In particular, there has been an emphasis on the development of high-throughput
methodologies to study the structural and functional aspects of glycan-protein interactions. The use of carbohydrate binding
proteins (i.e. lectins) in a microarray format has greatly enhanced our ability to de-convolute the structural aspects of
the glycome. This simple and unique technology provides a rapid method for glycomic analysis, which opens up the field of
glycobiology to more systems-based approaches towards function.
KeywordsLectin-Glycan-Glycomics-Glycosylation-Carbohydrate analysis-Microarray
12/2010: pages 91-102;
-
ChemBioChem 06/2010; 11(9):1203-7. · 3.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Carbohydrates are the most difficult class of biological molecules to study by high-throughput methods owing to the chemical similarities between the constituent monosaccharide building blocks, template-less biosynthesis, and the lack of clearly identifiable consensus sequences for the glycan modification of cohorts of glycoproteins. These molecules are crucial for a wide variety of cellular processes ranging from cell-cell communication to immunity, and they are altered in disease states such as cancer and inflammation. Thus, there has been a dedicated effort to develop glycan analysis into a high-throughput analytical field termed glycomics. Herein we highlight major advances in applying separation, mass spectrometry, and microarray methods to the fields of glycomics and glycoproteomics. These new analytical techniques are rapidly advancing our understanding of the importance of glycosylation in biology and disease.
Annual Review of Analytical Chemistry (2008) 06/2010; 4:367-92. · 9.05 Impact Factor
-
Current opinion in chemical biology 10/2009; 13(4):373-4. · 8.30 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Carbohydrates encode biological information necessary for cellular function. The structural diversity and complexity of these sugar residues have necessitated the creation of novel methodologies for their study. This review highlights recent technological advancements that are starting to unravel the intricate web of carbohydrate biology. New methods for the analysis of both glycoconjugates and glycan structures are discussed. With the use of these innovative tools, the field of glycobiology is poised to take center-stage in the postgenomic era of modern biology and medicine.
ACS Chemical Biology 10/2009; 4(9):715-32. · 6.45 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Glycosylation, a ubiquitous post-translational modification of proteins and lipids that generates enormous functional diversity, is rapidly gaining attention in the postgenomic era. The systematic study of glycans, that is glycomics, has been driven by the development of new analytical tools well suited to the inherent complexities of carbohydrate analysis, such as lectin-based microarray technologies. Recent work has demonstrated the utility of these analytical tools for glycomics in both clinical and research settings, for example identifying novel biomarkers associated with disease progression or studying HIV-1 exit mechanisms. This review highlights these new lectin-based microarray technologies and their role in the emerging field of glycomics.
Current opinion in chemical biology 09/2009; 13(4):427-32. · 8.30 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: HIV-1 is a master at deceiving the immune system and usurping host biosynthetic machinery. Although HIV-1 is coated with host-derived glycoproteins, only glycosylation of viral gp120 has been described. Here we use lectin microarray technology to analyze the glycome of intact HIV-1 virions. We show that the glycan coat of human T cell line-derived HIV-1 matches that of native immunomodulatory microvesicles. The carbohydrate composition of both virus and microvesicles is cell-line dependent, which suggests a mechanism to rapidly camouflage the virus within the host. In addition, binding of both virus and microvesicles to antiviral lectins is enriched over the host cell, raising concern about targeting these glycans for therapeutics. This work also sheds light on the binding of HIV-1 to galectin-1, an important human immune lectin. Overall, our work strongly supports the theory that HIV-1 co-opts the exocytic pathway of microvesicles, thus potentially explaining why eliciting a protective antiviral immune response is difficult.
Nature Chemical Biology 03/2009; 5(4):244-50. · 14.69 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Glycomics, i.e. the high-throughput analysis of carbohydrates, has yet to reach the level of ease and import of its counterparts, genomics and proteomics, due to the difficulties inherent in carbohydrate analysis. The advent of lectin microarray technology addresses many of these problems, providing a straightforward approach for glycomic analysis. However, current microarrays are limited to the available lectin set, which consists mainly of plant lectins isolated from natural sources. These lectins have inherent problems including inconsistent activity and availability. Also, many plant lectins are glycosylated, complicating glycomic evaluation of complex samples, which may contain carbohydrate-binding proteins. The creation of a recombinant, well-defined lectin set would resolve many of these issues. Herein, we describe an efficient strategy for the systematic creation of recombinant lectins for use in microarray technology. We present a small panel of simple-to-purify bacterially-derived lectins that show reliable activity and define their binding specificities by both carbohydrate microarray and ELISA. We utilize this panel to create a recombinant lectin microarray that is able to distinguish glycopatterns for both proteins and cell samples. This work opens the door to the establishment of a vast set of defined lectins via high-throughout approaches, advancing lectin microarray technology for glycomic analysis.
Molecular BioSystems 07/2008; 4(6):654-62. · 3.53 Impact Factor
-
Lara K Mahal
[show abstract]
[hide abstract]
ABSTRACT: Cell surface glycoconjugates control a variety of biological events including cell differentiation, homing to specific tissues, cell adhesion, virus/cell recognition and immunological recognition. The heterogeneity and diversity of these molecules present a challenge to understanding both their functions and how those functions are encoded. Advances in biotechnology have led to new methods for genome and proteome study that allow for the analysis of the entire genetic or protein content of a cell. Efforts towards the systematic study of the variations in cellular glycoconjugates are, in contrast, in their infancy. Recent advances in glycosylation related technology have begun to open up the possibility of exploring both the structure and the functions of the glycome in a systematic manner. This review focuses on the latest mass spectrometry, microarray-based and computational technologies for glycomics.
Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Anti-Cancer Agents) 02/2008; 8(1):37-51. · 2.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Glycosylation creates an intricate and complex code for biological information that plays a role in cell-cell communication, infection, and immunity among many biological events. Dynamic changes in the glycosylation status of cells have been observed in tumor cell metastasis and cell differentiation but have been difficult to analyze because of a lack of high-throughput and facile technologies. Here, we present a method for the rapid evaluation of differences in the glycosylation of heterogeneous mammalian samples using a ratiometric two-color lectin microarray approach. This work represents a significant improvement in glycomics technology and sets the stage for the systematic evaluation of how glycans encode biological information in complex systems.
Proceedings of the National Academy of Sciences 08/2007; 104(28):11534-9. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Carbohydrates coat most types of cell in nature and are intimately involved in various biological events, including cell differentiation, homing to specific tissues, cell adhesion, cell recognition, microbial pathogenesis and immunological recognition. Carbohydrate structures are complex to analyze owing to their branched nature, the diversity of secondary modifications of monomers, their indirect relationship to the genome and the range of molecular contexts in which the modifications are found. Thus, whereas the fields of genomics and proteomics have become accessible to most scientists, technologies to assess glycan structures rapidly (i.e. glycomics) are still in the developmental stages. This review focuses on recent developments in glycomic technologies, including new high-throughput techniques for glycan purification and annotation that are advancing mass-spectrometry-based glycomics, and the latest work on microarray methodologies to decipher the glycome.
Current Opinion in Chemical Biology 07/2007; 11(3):300-5. · 9.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Glycosylation is one of the most common posttranslational modifications, with more than half of all known proteins thought to be glycoproteins. Alterations in glycosylation play a role in a diverse set of biological phenomena including tumor cell metastasis, intracellular communication, and inflammation. The complexity of glycosylation at the molecular level and the lack of rapid analytical tools complicate the study of glycan function. We have recently developed a lectin microarray for the high-throughput analysis of glycosylation. Lectins are carbohydrate-binding proteins that have been used for decades as a detection method for glycans. By placing the lectins in a microarray format and using standard microarray printing and scanning technology, we have created a simple yet powerful technique for glycan profiling.
Methods in molecular biology (Clifton, N.J.) 02/2007; 385:193-203.
